An important webinar for muscle researchers discussing characterization of complete muscle function by combining lengthening, shortening and isotonic contraction tests with traditional isometric twitch and tetanus measurements. Quantifying muscle function continues to be an important part of any research where muscle is being directly or indirectly studied. However, a broad survey of literature reveals research remains heavily skewed towards isometric twitch and tetanus testing, which may not necessarily provide the most physiologically relevant data to the researcher. This webinar intends to discuss the subject of complete characterization of muscle and how scientists can combine a number of functional tests in their experimental design to better reveal scientific findings relating to muscle dynamics. In this webinar, sponsored by Aurora Scientific, experts will show how to increase your experimental toolbox to create protocols that utilize lengthening, shortening and isotonic contractions, in addition to the more common isometric tests. Attendees will also learn when best to implement these new protocols to obtain the most complete data possible.

1. Beyond Isometric Twitch:
Utilizing lengthening, shortening and isotonic contraction
tests for muscle function research.
Matt Borkowski
Aurora Scientific
Robert W. Grange, PhD
Virginia Tech
Sponsored by:

2. InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.

3. Thank you to our event sponsor
Aurora Scientific, a trusted
provider of instrumentation
for research in muscle
physiology, neuroscience
and material science.

4. Utilizing lengthening, shortening
and isotonic contraction tests for
muscle function research.
Matt Borkowski
Sales & Support Manager
Aurora Scientific
Copyright 2015 M. Borkowski, Aurora Scientific & InsideScientific. All Rights Reserved.

5. THE JOURNEY OF A THOUSAND MILES
BEGINS WITH A SINGLE STEP
-- Lao Tzu

6. • Aurora has served the muscle
community for nearly 20 years.
• Test systems and solutions ranging
from single cells up to the whole
animal.
• Friendly, reliable support.
Cell
Whole
Animal
Fiber
Whole
Muscle
About Aurora Scientific

7. A NEW CHAPTER BEGINS…

9. • Aurora Scientific believes in
providing solutions for the
complete characterization of
muscle.
• To completely characterize
the muscle, the 3 types of
contractions must be used.
Complete
Characterization
concentric
isometric
eccentric
What is complete characterization?

10. What is complete characterization?
• Isometric: Contraction at a
constant muscle length.
• Eccentric: Contraction while
the muscle is lengthening.
• Concentric: Contraction while
the muscle is shortening (can
be isotonic).
Complete
Characterization
concentric
isometric
eccentric

11. Strengths
 Simple, standardized protocols which
can be used across an array of muscles.
 Excellent way of assessing the absolute
strength of most muscle types.
 Useful for studying the basic
mechanisms behind contraction and
relaxation.
Challenges
Isometric Strengths
 Not the most physiologically relevant
model.
 Limited amount of information can be
obtained from the contractile data.

12. Strengths
 A good protocol to study muscle injury
and damage and create conditions to
study recovery.
 Excellent for inducing hypertrophy
when applied as an in-vivo training
protocol.
 Models exercise much more accurately
than isometric.
Eccentric Strengths
 A great deal of passive tension is
generated; may necessitate multiple
transducers if studying a broad array of
animal models and muscle types.
 Determining the correct protocol for
specific muscle types and animal
models can be a challenge.
Challenges

13. Strengths
 Offers a wealth of information within the
contractile data: Power, Work, Force-
Velocity relationship can all be measured.
 Isotonic protocols closely mirror real life
work and exercise.
 Either force or velocity can be controlled,
allowing for multiple ways to test a
particular hypothesis.
Concentric Strengths
 Can be technically challenging to
implement.
 Requires a good deal of configuration
for different muscle types.
Challenges

14. How Does
it Work ?

15. • Dual Mode Lever System allows for complete
characterization.
• Single instrument: a motor and force transducer
in one.
• Motor controls and senses position; Force is de-
convolved in electronics from motor current
signal.

16. • Lever systems range in size to accommodate small
cardiac muscles to limb muscles from larger
mammals.
• Single attachment point opens up the possibility of
performing different assays.
• Lever systems are often paired with experimental
chambers, apparatus and software.

17. One attachment point: 3 experiment types
In vitroIn situIn vivo
Click Here to see specific Aurora
Muscle Physiology Apparatus

18.  The foot is secured in a foot-plate mounted
to the dual mode lever system.
 Percutaneous or subcutaneous electrodes
can elicit muscle contraction.
 Aggregate torque of the plantar or dorsi
flexors of the lower limb can be measured.
 Resistance of the pedal can be adjusted to
create isotonic resistance.
 Foot pedal can rotate in conjunction with
contraction to create eccentric or
concentric conditions.
In vivo –
Ankle Torsion
Courtesy of Yan lab, UVA

19. Courtesy of Granzier lab, Arizona
• The hind limb is stabilized and the muscle of
interest revealed by surgically removing skin.
• The muscle of choice is partially dissected and
the one exposed tendon is tied to the dual
mode lever system.
• Direct muscle or nerve stimulation will produce
a muscle contraction which can be synchronized
with the lever system.
• Technique opens up the possibility to fully
characterize muscles without two easily
accessible tendons.
In situ –
One Free Tendon

20. • Muscle dissected from animal and
sutured at both free tendons.
• Muscle activated via field stimulation.
• Classical, vertical bath configuration
means only one tendon can attach to
an instrument.
• Only the dual mode lever system
permits tension & length to be
recorded and controlled in this
orientation.
In vitro –
Isolated Muscle
Courtesy of Barton Lab, UFL

21. Who can benefit from going
beyond isometric?
 Muscle Physiologists
 Exercise Scientists
 Bioengineers & Biologists
 Metabolic & Cardiovascular
Scientists
 Geneticists
 Neuroscientists
 Pharmacologists & Biochemists
 Anyone studying muscle
mechanics

22. Isometric and Dynamic Muscle
Function Assessment
Robert W. Grange, PhD
Department of Human
Nutrition, Foods and Exercise,
Virginia Tech
Copyright 2015 R.W Grange, Aurora Scientific & InsideScientific. All Rights Reserved.

23. When muscle is changed by…
Training
Disease
Drug
Genetic manipulation
Other…
Does function change in a meaningful way?

24. Skeletal Muscle Function in vitro

25. Patient Prep
‘Hang’ EDL muscle
Operating Room
‘Dissect’ EDL Muscle

26. Servomotor and
Isometric Force
Transducers
Dual-mode Servomotor For
Dynamic Contractions
Isometric ForceTransducer
Stepper Motor For Maintaining L0
Muscle Clamps

27. - +
EDL
Isometric and Dynamic
Contractions
Basic muscle in vitro preparation
1. Muscle is mounted in a bath
• Clamped at bottom
• Secured to motor arm at top
2. Electrodes activate muscle
3. Servo Arm
• Stays horizontal (isometric contraction)
• Moves up (eccentric contraction)
• Moves down (concentric contraction)
• Eccentric and concentric are dynamic
contractions

28. Torque = Force x Moment arm
Ma
Force
Skeletal Muscle
Function in vivo
Mouse Hindlimb

29. Torque = Force x Moment arm
Ma
Force
Skeletal Muscle
Function in vivo
Dog Hindlimb

30. Skeletal Muscle
Fundamentals

31. Epstein M, Herzog W.. Philos Trans R Soc Lond B Biol Sci 2003;358:1445-1452.
Anatomy
• Muscle
• Fascicles
• Fibers
• Myofibrils
• Sarcomere (functional unit of muscle)
• Z lines
• Myosin and Actin: contraction

32. Muscles: fibers classified by contractile and biochemical properties. Typically Use: Extensor digitorum longus (EDL) – fast ; Soleus – slow
Skeletal Muscle Fiber and Muscle Types
Properties
Fiber Type Classification
Slow-Oxidative (SO) Fast-Oxidative-Glycolytic (FOG) Fast-Glycolytic (FG)
Predominant MHC Type I Type IIA Type IIX or IIB
Contractile Velocity Slow Intermediate High
Glycolytic Potential Low Intermediate High
Oxidative Potential High Intermediate Low
Mitochondrial Density High Intermediate Low
Myoglobin Content High Intermediate Low
Resistance to Fatigue High Intermediate Low

33. DHPRRyR
Excitation – Contraction
Coupling
• Excitation: Action potential…
• Via T-tubule…
• Releases calcium from RYR of
Sarcoplasmic Reticulum…
• Calcium binds Troponin…
• Myosin and Actin interact:
• Contraction

34. -
dF/d
t
(Rest
force) Lo
(Peak
force)
Active
force
Time to Peak Force
(TPF)
Half- Relaxation
Time (HRT)
Isometric Twitch
A twitch is the
contractile
response to a single
Action Potential or
single (1 Hz)
electrical
stimulation

35. Force Summation

36. t peakF base
Peak Force
(Rest Force)
Isometric Maximal Tetanus
A maximum tetanus
is the result of
maximal summation:
no further increase in
force output despite
increased frequency
of activation.

37. 1 30 50 80 100 150
Frequency (Hz)
0
5
10
15
20
25
30
35
Stress(g/mm
2
)
- Control
- MDX
- MDX:U
-/-
*
*
*
*
*
*
†
†
Stress = force (g)/muscle cross sectional area (mm2); mN/mm2
EDL Muscles
Force Summation
Stress-Frequency
• Stress = force/muscle
cross sectional area
• Stress increases with
increased activation
frequency (i.e.,
summation)
• The maximum stress is
the maximum tetanus

38. 0.00 0.20 0.40 0.60 0.80 1.00
Fractional Load (F/Fat)
0.0
2.0
4.0
6.0
8.0
10.0
ShorteningVelocity(Lf/s)
* *
*
** * * * * * * *
25%
50%
75%
Fractional Load
Tetanic Afterload Method
F at = maximum tetanic Force
Vmax
EDL
Soleus
Force-Velocity

39. EDL
Soleus
Power –
FxV; Work/Time
• Peak power typically
occurs at a fractional
load of 0.40 (40%) of
maximum load

40. EDL vs Soleus Muscle Fatigues More Quickly

41. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

42. Increased extraocular muscle strength with direct injection of insulin-like growth factor-I.
Anderson, Christiansen, Grandt, Grange, McLoon. Invest. Opthalmol Vis Sci 47(6):2461-7,
2006.
CONCLUSIONS:
Direct muscular injection of IGF-I
effectively increases EOM force
generation without the potential
biomechanical hazards of surgery
such as permanently altered muscle
length or insertional position on the
globe.
• 25 ug IGF-1
• rabbit superior rectus
muscle
• assess after 1 week
Increased Extraocular
Muscle Strength

43. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

44. Concerted regulation of myofiber-specific gene expression and muscle performance by the transcriptional repressor Sox6.
Quiat, Voelker, Pei, Grishin, Grange, Bassel-Duby, Olson. PNAS 108(25):10196-201, 2011
Inhibition of Sox6 leads to a fast to slow phenotype shift
Sox6 - a transcriptional repressor of slow fiber phenotype

45. (Quiat 2011)
1. Increased red color
Fast to Slow Fiber Shift…
2. Decreased fiber cross sectional area

46. Force-Velocity
relationship:
Sox6 KO EDL has
decreased Vmax
Fatigue protocol:
Sox6 KO EDL and
Soleus fatigue
less
Muscle Function Changes With Sox6 Knockout

47. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

48. SarcolemmaDGC
Ervasti, J.M., J.Biol.Chem.,2003. 278(16): p. 13591-13594.
Fiber DGC Absent From
Sarcolemma:
Dystrophic muscle
membrane more
“leaky”
Duchenne Muscular Dystrophy

49. - +
EDL
Procion Orange
(Mr 631)
Stretch – Injury Protocol
Fiber

50. 0.00 0.25 0.50 0.75 1.00 1.25 1.50
Time (s)
0.0
1.5
3.0
4.5
6.0
7.5
Force(g)
isometric
0.1 Lo stretch
Rate:0.5Lo/s
500 ms 200 ms
Stretch Protocol
• Muscle contracts
isometrically for 500ms
• Then is stretched for 200
ms while contracting
• Stimulation ends at the
peak of the stretch
• The muscle relaxes.

51. Fast-twitch skeletal muscles of dystrophic mouse
pups are resistant to injury from acute mechanical
stress. Grange, Gainer, Marschner, Talmadge, and Stull.
Am J Physiol Cell Physiol 283(4):C1090-101, 2002.
No
stretch
Five
stretches

52. Uptake of dye by EDL during stretch injury protocol
in mdx mice aged 4 months ~23%
Fast-twitch skeletal muscles of dystrophic mouse
pups are resistant to injury from acute mechanical
stress. Grange, Gainer, Marschner, Talmadge, and Stull.
Am J Physiol Cell Physiol 283(4):C1090-101, 2002.

53. - +
EDL
Stretch Injury Protocol:
test microdystrophin gene
therapy in mdx mice

54. Adeno-associated virus-
mediated microdystrophin
expression protects young
mdx muscle from
contraction-induced
injury.
Liu, Yue, Harper, Grange,
Chamberlain and Duan.
Mol. Ther. 1(2):245-56 2005.
microdystrophin revertant dystrophin
treated
untreated

55. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

56. Stiffness – change in
force during a change in
muscle length
• for stress-strain
assessment of achilles
tendon to determine
stiffness in mouse pup
aged 15 days
• Grange Lab 2-12-2013
Hindlimb Prep

57. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

58. - +
EDL
P1 Hindlimb
Force Assessment in
Mouse Pup Hindlimbs

59. KLHL40 deficiency
destabilizes thin
filament proteins and
promotes nemaline
myopathy
Ankit Garg,1 Jason O’Rourke,1 Chengzu
Long,1 Jonathan Doering,2 Gianina
Ravenscroft,3 Svetlana
Bezprozvannaya,1 Benjamin R.
Nelson,1 Nadine Beetz,1 Lin Li,4 She
Chen,4 Nigel G. Laing,3 Robert W.
Grange,2 Rhonda Bassel-Duby,1 and
Eric N. Olson1 J Clin Invest.
2014;124(8):3529–3539.

60. A. P8 diaphragm stained with desmin (red); DAPI
(blue) and wheat germ agglutinin (green)
B. EM of longitudinal sections of P8 diaphragm
KLHL40 KOs Have
Disrupted Sarcomeres
Tetanic force
response of P1
hindlimb
Is function disrupted at ages
earlier than P8?
10 mm
EDL

61. Examples of Muscle Function
Assessment
1. IGF-1 Injection Into Extraocular Muscle
2. Sox6 Knock Out Mouse
3. Duchenne Muscular Dystrophy (Stretch Injury Protocol)
4. Mechanical Properties – Achilles Tendon stiffness
5. P1 mouse hindlimb in vitro
6. Mouse/dog hindlimb in vivo

62. Mouse Plantar Flexor
Torque in vivo
Torque = Force x Moment arm
Ma
Force

63. Age- 9 weeks Age- 17 weeks
The Advantage
of In Vivo:
Assess muscle
function over
time between
conditions
Age- 21 weeks Age- 25 weeks

64. Dog Plantar Flexor
Torque in vivo
Torque = Force x Moment arm
Ma
Force

65. Eccentric contractions
induce rapid isometric
torque drop in
dystrophin-deficient
dogs.
Tegeler, Grange, Bogan,
Markert, Case, Kornegay,
Childers. Muscle Nerve
42(1):130-2, 2010.
Dog In Vivo #1: Dystrophic Dog Hindlimb Muscle Function

66. Gene Therapy Prolongs Survival and Restores Function
in Murine and Canine Models of Myotubular Myopathy
Martin K Childers1,2,†, Romain Joubert3, Karine Poulard3, Christelle Moal3,
Robert W Grange4, Jonathan A Doering4, Michael W Lawlor5,6, Branden E.
Rider5, Thibaud Jamet3, Nathalie Danièle3, Samia Martin3, Christel Rivière3,
Thomas Soker6, Caroline Hammer3, Laetitia Van Wittenberghe3, Mandy
Lockard7, Xuan Guan7, Melissa Goddard7, Erin Mitchell7, Jane Barber7, J. Koudy
Williams7, David L Mack1, Mark E Furth8, Alban Vignaud3, Carole Masurier3,
Fulvio Mavilio3, Philippe Moullier3,9,10, Alan H Beggs5,†, and Anna Buj-Bello3,†
Sci Transl Med. 2014 January 22; 6(220): 220ra10. doi:10.1126/scitranslmed.3007523.
Dog In Vivo #2: MTM-Deficient Dog Hindlimb
Muscle Function

67. “Loss-of-function mutations in the myotubularin gene
(MTM1) cause X-linked myotubular myopathy (XLMTM),
a fatal, congenital pediatric disease that affects the entire
skeletal musculature.” Childers et al., 2014
Dr. Childers (University of Washington) has a colony of XLMTM dogs.
MTM1 encodes a lipid phosphatase; primarily effects skeletal muscle:
• Hypotrophic fibers
• Muscle structural abnormalities
• Generalized weakness
There is no known cure… what are the functional outcomes of gene therapy?

68. Childers et al., Sci Transl Med. 6(220), 2014
Hindlimb infusion of XLMTM
dogs with AAV8-MTM1 (canine)
AAV-infused Non-infused
VL
CT
Baseline age: 9 wks
6 wks post-
inf
8 wks post-
inf
14 wks
post-inf
1 year
post-inf

69. Acknowledgements
The authors and co-authors listed herein: thank you for
providing me the opportunity to contribute to your great work!
Audentes
Therapeutics

70. ?
Muscle function:
Physiological
Interpretation is
Essential (PIE)
PIE

71. Robert W. Grange, PhD
Virginia Tech
rgrange@vt.edu
Matt Borkowski
Aurora Scientific
mattb@aurorascientific.com
Thank You!
For additional information on Aurora
Scientific instruments specially designed
for muscle function research please visit:
http://www.aurorascientific.com/